Tumor cell heterogeneity and physiological variability are two important characteristics which significantly affect the response of tumors to therapeutic intervention. However, little is known about the heterogeneous response of malignant cells to X-irradiation. Thus, the long term goal of this application is the development of a more complete understanding of the molecular and metabolic phenomena which underlie the heterogeneous cellular responses to X-irradiation. This is a most complex task in vivo; thus, the heterogeneous murine mammary carcinoma cell lines designated 66 & 67, growing in monolayer culture will be utilized. Specific aims include: 1) the measurement of the clonogenicity (cell survival) of 66 vs. 67 cells after X-irradiation in the following physiological states: active proliferation (P, i.e., exponential growth), transition (T, P-cells moving towards quiescence), quiescent (Q, nonproliferating, unfed plateau culture), and stimulated (refeded Q cells (St); 2) the determination of X- ray-induced DNA damage and repair for the cells in Aim 1. The alkaline filter elution, alkaline sucrose gradient and nucleoid sedimentation assays will be used; 3) the documentation of the degree of condensation of chromatin as defined by nuclease digestion assayed by radiochemical techniques and polyacrylamide gel electrophoresis; 4) the characterization of the "thiol status" and the "poly(ADP-ribosyl)ation status" of the cells defined in Aim 1. This will be accomplished by biochemical (colorimetric) assays for total cellular protein sulfhydryls, nonprotein sulfhydryls, and glutathione. Also, biochemical/radiochemical assays will be used to measure cellular levels of NAD and poly(ADP-ribose) synthetase, and polyacrylamide gel electrophoresis will be used to evaluate the differential extent of poly(ADP-ribosyl)ation of the nuclear proteins; and 5) comprehensive correlation analyses of the results from the physiological and molecular studies with the cellular response of the 66 and 67 cells. Thus, these studies will use the heterogeneous cell model system to demonstrate: 1) the relationship between X-ray induction of DNA damage and repair and X-ray cytotoxicity under various physiological states; 2) the role of the chromatin state on DNA damage and repair; 3) the role of endogenous thiols (radical scavengers) as a modifier of the molecular and cellular radiation response, and 4) the role of poly(ADP-ribosyl)ation on the molecular and cellular radiation response, and finally 5) which if any, of these mechanisms explain, in part, the cellular heterogeneous responses to X-irradiation.